Our long-term goal is to develop genetic approaches that could be used for the treatment of mitochondrial disorders associated with mitochondrial DNA (mtDNA) mutations. We propose to develop methods that improve oxidative phosphorylation function in cells harboring heteroplasmic mtDNA mutations (i.e. a mixture of wild-type and mutated mtDNA). Our approach is based on the mtDNA heteroplasmy shift concept, where, by genetic manipulation, the levels of the mutated genome are decreased in relation to the wild-type genome, thereby improving cellular health. In the previous funding period we showed that mitochondria-targeted restriction endonucleases are powerful tools to achieve mtDNA heteroplasmy shift. The present proposal will further expand this approach to any mtDNA mutation. To do so, we will take advantage of new developments in the field of designed nucleases. TAL-effector nucleases have emerged as highly flexible molecules that can recognize DNA regions based on a modular arrangement of their DNA binding domains. We have obtained preliminary data showing that this approach is feasible and propose to better characterize the system in vitro, ex vivo and in vivo.